Splenic lymphocytes from young, 15 month old and 24-28 month old C57BL/6 mice housed in a constant environment were studied. Single cell suspensions were cultured in vitro with T cell mitogens, phytohaemagglutinin and Concanavalin A, and the B cell mitogen, lipopolysaccharide. Functional capacity of the T and B lymphocytes was assessed by the mitogen-induced incorporation of tritiated thymidine by dividing cells. Age-related changes in the properties of circannual rhythms expressed by T and B lymphacytes following activation by mitogens in vitro were described previously and more critical evaluations of some parameters have been made. Phase reference points calculated for each data series showed increases in free-running periods of cells stimulated with Concanavalin A from 371 days for young mice to 458 days and 455 days for 15-month old and senescent mice, respectively. Similar increases with age in periods by an average of 55 days and 168 days for cells activated with phytohemagglutinin and lipopolysaccharide were found. Power spectral analysis of each data series and analyses for coherence between pairs of series provided independent, objective evidence for similar low frequency rhythms (longer than 52 weeks) in all data series. These changes in periodicity in addition to decreased amplitudes of T but not B cell rhythms in cells from senescent mice result in another example of imbalance in the immune system with age. The degree of stress induced by cryopreservation of lymphocytes from young mice changed seasonally and was maximal during the months that activation of unfrozen cells was minimal. During the fall and winter, decreased percentages of viable cells were recovered, greater numbers of cells lysed, and mitogenic stimulation in vitro resulted in reduced incorporation of tritiated thymidine. These seasonal patterns of change in cryopreservation properties resembled and reinforced the circannual rhythmicity in funcitons of unfrozen lymphocytes.